Invasive Ductal Carcinoma Cells Research Articles

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

Increase delivery, cytotoxicity, stability and induction of apoptosis in cisplatin by establishing a new complex with methotrexate

Cisplatin, as an effective anti-cancer drug, is widely used to treat breast cancer. However, undesirable side effects and drug resistance have partially limited its use for some patients. One of the causes of side effects is the lack of differentiation between cancer cells and normal cells, which could be reduced by targeted drug delivery. Therefore, we decided to replace methotrexate, as a targeting ligand, with chlorine atoms of cisplatin and evaluate its effect on cytotoxicity and induction of apoptosis in T47D (invasive ductal carcinoma) cell lines. FTIR and 13C NMR methods proved the coordination of methotrexate to cisplatin and the formation of the eight-membered ring between them. The results of the MTT assay showed that the cis-[Pt(NH3)2MTX] complex showed a time- and dose-dependent cytotoxic effect at both periods of 48 and 72 h. Although the IC50 of the new complex was higher when compared to cisplatin (78 vs. 33 μM), it completely inhibited the cell viability of the T47D cells after 72 h of exposure. The results of the real-time PCR test approved that the cis-[Pt(NH3)2MTX] complex can induce the intrinsic apoptosis pathway by increasing the level of Bak1 proteins, while cisplatin does not affect any of the studied genes. Molecular docking confirmed that four amino acids of folate receptors such as ASP 81, SER 174, SER 101, and TRP 138 were involved in binding to the cis-[Pt(NH3)2MTX] complex. Finally, the atoms-in-molecules method and molecular electrostatic potential confirmed that the new complex has more stability than cisplatin via the coexistence of the Intra-HB and Inter-HB interactions.

Comparative study of microscale and macroscale technique for encapsulation of Calotropis gigantea extract in metal-conjugated nanomatrices for invasive ductal carcinoma

The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV–Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments.

Abstract 3645: OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression

Abstract Ductal carcinoma is the most commonly diagnosed breast cancer in women, the presence of metastases drops patient five-year survival from 95% to an abysmal 27%. This makes studying the mechanisms that promote metastasis of critical importance. Research shows that the extracellular matrix (ECM) of the tumor microenvironment (TME) plays an important role in invasive ductal carcinoma (IDC) progression and metastasis. Specifically; the density, stiffness, and orientation of collagen I fibers in the stroma all impact IDC motility, invasion, and metastasis. Lysyl oxidase-like 2 (LOXL2) is a secreted enzymatic protein that is implicit in ECM remodeling. By catalyzing an oxidative reaction in peptidyl lysine/hydroxylysine present on collagen I fibers, LOXL2 initiates crosslinking of the fibers. Our previous publication demonstrates that interleukin-6-related pro-inflammatory cytokine signaling, specifically oncostatin M (OSM), led to the overexpression and secretion of the LOXL2 enzyme. This increased the metastatic potential of IDC cells by promoting the crosslinking and alignment of collagen I fibers, which in turn led to increased invasion in 3D collagen I matrices. Thus, we hypothesize that OSM-induced LOXL2 expression, secretion, and subsequent collagen I fiber crosslinking and alignment, leads to an increase in IDC metastasis. In vivo studies utilizing nude athymic mice injected with either MCF7-Luc-EV control or MCF7-Luc-OSM overexpressing cells were performed in conjunction with LOXL2 inhibition using specially designed chow containing either LOXL2 SMI or control. Further analysis of OSM signaling pathways was performed in MCF7 and MDA-MB-468 IDC cells to determine mechanism for OSM induction of LOXL2. Our studies demonstrate that canonical OSM signaling pathways (pERK, pSTAT3, and pAKT) all contribute to LOXL2 expression in MCF7 cells suggesting a possible mediator protein we identified as c-Myc. Furthermore, OSM-induced LOXL2 played a significant role in OSM promoted tumor growth and metastasis as LOXL2 inhibition led to decreased tumor size and far less metastatic lesions. We also analyzed and confirmed a synergistic interaction between OSM and interleukin-1beta (IL-1β) in LOXL2 expression. These results confirm the significance of LOXL2 expression in OSM promoted metastasis and suggests LOXL2 would be an important target for future breast cancer therapies, specifically in patients with elevated levels of OSM. Furthermore, the synergistic interaction between OSM and IL-1β in LOXL2 expression suggests that patients with high levels of both cytokines are at even greater risk of metastasis, making these great markers for determining at risk IDC patients. Citation Format: Simion Dinca, Cody Wolf, Laura Bond, Cheryl Jorcyk. OSM-induced LOXL2 expression hinges on upregulation of c-Myc and promotes tumor growth and metastasis, IL-1beta plays synergistic role in LOXL2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3645.

Abstract PD10-02: PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms

Abstract Breast cancer is a leading cause of female mortality and despite advancements in diagnostics and personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. The druggable estrogen receptor (ER, ESR1), overexpressed in two-thirds of all breast cancer, evolves in 30% of tumors exposed to endocrine therapy consequently resulting in treatment resistance. A more recently discovered mechanism of ER mediated endocrine resistance is ER fusion proteins. ER fusions, found predominately in metastatic endocrine resistant disease, harbor ESR1 exons 1-6 fused to an in-frame gene partner due to an ESR1 intron 6 translocation break. Our lab has demonstrated that ER fusion proteins, which lack the C-terminal ligand-binding domain (LBD), recapitulate phenotypes of ER proteins harboring endocrine-resistant point mutations occurring in the LBD. Our research goals aim to 1) determine fusion prevalence and emergence, 2) understand fusion mechanisms of resistance and 3) explore alternative treatment options for our patients. The promiscuous nature of fusion partners hinders fusion detection by traditional sequencing and thus our research team has developed a novel ER fusion detection method, EnRich. The EnRich probe set (4,324 probes in total) is composed of two separate probe pools targeting at 2x tiling intronic and exonic ESR1, the upstream promoter region and select oncogenic genes. To optimize the EnRich pipeline, DNA was extracted from a frozen tumor sample and a PDX model harboring ESR1-DAB2 and ESR1-LPP fusions, respectively. ESR1-DAB2 and ESR1-LPP were accurately detected by quantifying discordant paired-end or split reads mapped to chromosome 6. In addition, liquid biopsies from 15 patients with ER positive advanced breast cancer were also assessed through EnRich to uncover unidentified ESR1 structural variants. Two patient samples were detected to harbor ESR1 fusions (ESR1-CCDC170, ESR1-AKAP12 and ESR1-YAP1) and were further validated in corresponding mRNA. These recurrent and novel fusions were supported with more than 10 reads each, indicating that the EnRich pipeline is an effective and accurate sequencing approach to understanding ER fusion prevalence. Our lab, furthermore, has studied ER fusion proteins mechanistically. We have stably overexpressed ER fusions in transgenic breast cancer cell lines engineered with shRNA targeting the endogenous wildtype ER (ESR1-WT). In this ESR1-WT depleted cellular context, we found that ER fusions (notably ESR1-SOX9 and ESR1-YAP1) demonstrate ER hyperactivation through an estrogen response element (ERE) assay compared to ESR1-WT and a truncated exon 1-6 ESR1 (ESR1∆CTD). Importantly, fusion ERE activity was robust in the absence of the ER ligand, estradiol, as well as in the presence of endocrine therapies, implying ER fusion proteins function in a ligand independent, endocrine resistant mechanism. ER fusion positive cell lines were also enriched in oncogenic phenotypes such as enhanced 3D growth, cell survival via colony formation, and migration in a wound scratch assay. These enhanced metastatic potentials of the ER fusions were observed in both invasive ductal and lobular carcinoma cell lines, albeit at varying magnitudes depending on the 3’ fusion partner and phenotype being assessed. Although the ER fusions harbored unique characteristics due to the C-terminal partner, transcriptomic profiling revealed that enhanced EMT and KRAS signaling signatures were shared among all fusions when compared to the ESR1-WT and ESR1∆CTD cell lines, which may serve as future exploitable drug targets. Comprehensive detection and functional evaluation of ER fusion proteins will provide clinicians and patients with better understanding of tumor endocrine-resistant prevalence and discovery of more effective treatment options. Citation Format: Megan E. Yates, Tiantong Liu, Jagmohan Hooda, Sichun Yang, Riyue Bao, Jennifer M. Atkinson, ADRIAN V. LEE, Steffi Oesterreich. PD10-02 Novel ER fusion detection method to gain insight in fusion prevalence and endocrine resistant mechanisms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-02.

The cyto-linker and scaffolding protein "plectin" mis-localization leads to softening of cancer cells.

Discovering tools to prevent cancer progression requires understanding the fundamental differences between normal and cancer cells. More than a decade ago, atomic force microscopy (AFM) revealed cancer cells' softer body compared to their healthy counterparts. Here, we investigated the mechanism underlying the softening of cancerous cells in comparison with their healthy counterparts based on AFM high resolution stiffness tomography and 3D confocal microscopy. We showed microtubules (MTs) network in invasive ductal carcinoma cell cytoskeleton is basally located and segmented for around 400 nm from the cell periphery. Additionally, the cytoskeleton scaffolding protein plectin exhibits a mis-localization from the cytoplasm to the surface of cells in the carcinoma which justifies the dissociation of the MT network from the cell's cortex. Furthermore, the assessment of MTs' persistence length using a worm-like-chain (WLC) model in high resolution AFM images showed lower persistence length of the single MTs in ductal carcinoma compared to that in the normal state. Overall, these tuned mechanics support the invasive cells to ascertain more flexibility under compressive forces in small deformations. These data provide new insights into the structural origins of cancer aids in progression.

Expression of NUCB2/NESF-1 in Breast Cancer Cells.

Recently, the expression of NUCB2/NESF-1 has been linked to tumor development. We report NUCB2/NESF-1 expression and its relation to clinicopathological parameters in breast cancer cells. Immunohistochemical reactions were conducted on 446 cases of invasive ductal carcinoma (IDC) and 36 cases of mastopathy. The expression of NUCB2/NESF-1 was also examined at the mRNA and protein levels in breast cancer cell lines. A statistically significant higher level of NUCB2/NESF-1 in IDC cells was noted compared to that in mastopathy samples. The level of NUCB2 expression in the cytoplasm of IDC cells decreased with the increasing degree of tumor malignancy (G). Higher NUCB2 expression was found in tumors with estrogen receptor (ER)-positive and progesterone receptor (PR)-positive phenotypes compared to that in estrogen-receptor-negative and progesterone-receptor-negative cases. Moreover, a higher expression was shown in ER(+) and PR(+) MCF-7 and T47D cell lines compared to that in triple-negative MDA-MB-468 and normal human breast epithelial cells. The analysis of the five-year survival rate indicated that a positive NUCB2/NESF-1 expression in tumor cells was also associated with longer patient survival. The study results suggest that NUCB2/NESF1 may play an important role in malignant transformation and may be a positive prognostic factor in IDC.

CK2α as a prognostic factor in invasive ductal carcinomas of the breast: cancer recurrence prognosis by surgical sampling

Breast cancer is the most common cancer in Japanese and Asian women. Although the 5-year survival rate of these patients is relatively high, recurrences and fatalities do occur. The objective of this work was to develop a reliable prognostic indicator to guide therapeutic intervention without waiting for recurrence or metastasis. We have previously shown that positive immunohistochemical staining of a protein kinase, CK2α (CSNK2A1), in nucleoli of invasive ductal carcinoma cells is strongly associated with tumour recurrence and poor patient outcomes. For this study, we did an immunohistochemistry investigation of the location of CK2α in the nuclei and nucleoli of formalin-fixed, paraffin-embedded tissue block samples from patients with invasive ductal carcinoma who underwent surgery in 2008-14 but no neoadjuvant chemotherapy at Hoshi General Hospital, Fukushima, Japan. Furthermore, a new study with 59 patients with luminal type breast cancer was done. Patients were followed-up (clinicopathological information and outcomes) for more than 5 years. To characterise CK2α-associated proteins systematically, nuclear and nucleolar extracts were prepared from MCF-7 cells and immunoprecipitated with anti-CK2α antibodies followed by nano-flow liquid chromatography mass spectrometry analysis. We did a Kaplan-Meier analysis to evaluate whether nucleolar CK2 staining might be associated with poor recurrence-free survival. This retrospective study was approved by the Institutional Review Boards of Fukushima Medical University and Hoshi General Hospital. Samples from 112 patients were included. 105 (94%) of 112 patients with invasive ductal carcinoma had high CK2α expression in nuclei, and 41 (37%) of 112 displayed it predominantly in nucleoli. Clinical and pathological malignancy was strongly correlated with high nuclear and nucleolar CK2α expression. Recurrence-free survival was significantly reduced in patients with positive nucleolar CK2α staining (n=41; median 7·89 years [IQR 4·86-8·86]), comparing with patients with negative nucleolar staining (n=71; median 8·89 years [7·87-9·79]; p<0·0001). Kaplan-Meier analysis showed a decrease of approximately 50% in recurrence-free survival in patients with nucleolar CK2α-positive stain in the triple-negative breast cancer (p=0·0069) and post-operative stage 3 (p=0·0073) groups. By contrast, no patients who relapsed or died in the triple-negative breast cancer group had negative staining for nucleolar CK2α. Evaluation of nucleolar CK2α can be considered as an independent prognostic factor because it reached significance in multivariate analysis (hazard ratio [HR] 5·26 [95% CI 1·35-20·55]; p=0·017). In addition, the HR of nucleolar CK2α-positive staining was less affected by adjustments in Cox proportional multivariate analysis. Cluster analysis showed that nucleolar CK2α-positive staining contributed more than any other clinicopathological factor to predicting the variation in the future length of survival. Analysis with luminal type breast cancer (n=59), in which the risk of recurrence is generally low and which is treated postoperatively with hormone therapy alone, showed that cancer recurrence and poor patient outcomes strongly associate with nucleolar CK2α-positive stain (n=7; p=0·037) by Kaplan-Meier analysis. In this case, nucleolar CK2α is considered a sole independent prognostic factor (HR 2·11 [95% CI 0·27-3·69]; p=0·0087). Evaluation of this method in other solid tumours is ongoing. Our results suggest that the evaluation of nucleolar CK2α-positive staining might be a new and independent prognostic factor that can improve treatment efficacy and patient quality of life after surgical resection of the tumour. The application of this analysis could contribute to early treatment strategy decisions and to companion diagnostics. The protein interactome of nuclear CK2 complexes showed CK2α's role in protein synthesis and RNA post-transcriptional modification. Specifically, a more detailed evaluation of CK2α function in nucleolar events in malignant cells might lead to the development of novel, more accurately targeted anti-cancer therapeutics. In conclusion, evaluating nucleolar CK2α staining in combination with other clinicopathological indicators might allow for a more accurate prediction of future recurrence or metastasis, which cannot be accomplished by current methods. Japan Agency for Medical Research and Development (grant number 20lm0203006j0004).

An exceedingly rare simultaneous incidental occurrence of synchronous primary malignancies; Invasive Ductal Carcinoma and Renal Cell Carcinoma in a male - A Case Report.

Introduction and importanceSynchronous malignancies are defined as the emergence of one or more tumors which either occur simultaneously or within 6 months of each other. Populations older than 50 years of age are the most vulnerable. Documented prevalence rates of synchronous neoplasia are 4.5–11.7 %. To the best of our knowledge, ours is the first documented case of synchronous primary incidental occurrence of Invasive Ductal Carcinoma (IDC) and Renal Cell Carcinoma (RCC) in a Middle Eastern male. This type of co-occurrence must be borne in mind because such neoplastic occurrence is potentially fatal. Documentation is essential to raise awareness and to decrease the resultant morbidity and mortality.Case presentationWe present a case of a 61-year-old male who presented to our clinic with a 22-day-history of gradual, painless, and disproportionate hypertrophy of his left breast. CT scan revealed incidental breast and right kidney masses. Therapeutic intervention included a modified radical mastectomy with Sentinel lymph node excision along with right radical nephrectomy.Clinical discussionTreatment of our patient was multimodal. Accurate radiological studying together with clinical examination helped us in making a diagnosis. Treatment options for this pathology consist of a combination of surgery and/or adjuvant therapy.ConclusionSynchronous IDC and RCC are an extremely rare co-occurrence, especially in males, particularly Middle Eastern males, and more specifically, those presenting asymptomatically as incidental findings. It is vital to further document and study such cases to establish innovative surgical techniques, screening modalities for males, and to overcome the consequential morbidity and mortality.

Expression of tumour transcription factor GLI1 in canine mammary tumours tissue.

BackgroundMammary tumor is one of the most common diseases of canine in pet clinics.ObjectivesThis study investigates the distribution and expression of the tumor transcription factor GLI1 and the downstream proteins, Bmi1 and Sox2, in canine mammary tumors and paracancerous tissues.MethodsCancerous and paracancerous normal mammary tissues were detected using western blotting (WB), and immunohistochemistry.ResultsThe results showed that the histopathology of different types in mammary tumors by microscopic observation. GLI1/Bmi1/Sox2 expression was significantly higher in canine mammary invasive carcinoma than in ductal carcinoma and adjacent normal mammary tissues (p < 0.01). The expression of GLI1 in invasive carcinoma tissues was significantly higher than Bmi1 and Sox2, while Sox2 expression in ductal carcinoma tissues was significantly higher than GLI1 and Bmi1 (p < 0.01). GLI1/Bmi1/Sox2 all showed positive reactions in both mammary tumor and adjacent normal mammary tissues with immunohistochemistry. GLI1 and Sox2 showed strong positive staining in the cytoplasm of invasive mammary carcinoma and ductal carcinoma cells, and weak positive staining in the nuclei. The positive Bmi1 reaction was mainly concentrated in the cytoplasm of invasive carcinoma and ductal carcinoma cells, while the positive reaction on the cell membrane was weak.ConclusionsWe speculate that GLI1 and related proteins play an important role in regulating the proliferation and differentiation of tumors. Therefore, it provides important reference for the pathogenesis and pathogenicity of canine mammary tumor.

Loss of E-cadherin Induces IGF1R Activation and Reveals a Targetable Pathway in Invasive Lobular Breast Carcinoma.

Overall, this suggests that these targets require further exploration in ILC treatment and that CDH1 loss may be exploited as a biomarker of response for patient stratification.

Classification and enhancement of invasive ductal carcinoma samples using convolutional neural networks

In the medical area, the detection of invasive ductal carcinoma is the most common sub-type of all breast cancers; about 80% of all breast cancers are invasive ductal carcinomas. Detection of this type of cancer shows a great challenge for specialist doctors since the digital images of the sample must be analyzed by sections because the spatial dimensions of this kind of image are above 50k × 50k pixels; doing this operation manually takes long time to determine if the patient suffers this type of cancer. Time is essential for the patient because this cancer can invade quickly other parts of the body. Its name reaffirms this characteristic, with the term "invasive" forming part of its name. With the purpose of solving this task, we propose an automatic methodology consisting in improving the performance of a convolutional neural network that classifies images containing invasive ductal carcinoma cells by highlighting cancer cells using several preprocessing methods such as histogram stretching and contrast enhancement. In this way, characteristics of the sub-images are extracted from the panoramic sample and it is possible to learn to classify them in a better way.

LINC00092 Suppresses the Malignant Progression of Breast Invasive Ductal Carcinoma Through Modulating SFRP1 Expression by Sponging miR-1827

Breast invasive ductal carcinoma (IDC) is a most common kind of breast cancer (BC), yet to date the corresponding effective therapies are limited. Extensive evidence has indicated that lncRNAs are involved in multiple cancers, and the potential mechanism of lncRNAs, such as LINC00092, mentioned in IDC remains elusive. IDC clinical samples from TCGA database were used to analyze the expression levels of LINC00092, miR-1827 and SFRP1. Kaplan-Meier method was applied to plot the overall survival curves. KEGG and GO were employed to screen the pathway that LINC00092 participated in. Pearson’s correlation analysis determined the relationship between LINC00092 and SFRP1. Bioinformatics analysis and dual-luciferase reporter assay examined the association among LINC00092, miR-1827, and SFRP1. Cell counting kit-8, colony formation and transwell assays were performed to detect cell viability, colony formation, and migration and invasion, respectively. Quantitative reverse-transcription polymerase chain reaction and western blot were utilized to investigate the expression at RNA and protein levels. LINC00092 expression was down-regulated in IDC tissues and cells, which was correlated with poor prognosis. Down-regulated LINC00092 facilitated cell proliferation, colony formation, and cell migration and invasion, while up-regulated LINC00092 inhibited cell malignant behaviors. LINC00092/SFRP1 physically bound to miR-1827 in IDC. SFRP1 expression was proportional to LINC00092 expression and inversely proportional to miR-1827 expression. The inhibitory effects of LINC00092 on cell aggressive behaviors were partially regulated by miR-1827/SFRP1. In summary, our results indicated that overexpression of LINC00092 inhibited the development of IDC through modulating miR-1827/SFRP1 axis, suggesting new therapeutic targets to treat IDC.

Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma.

Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.

Sharp Downregulation of Hub Genes Associated With the Pathogenesis of Breast Cancer From Ductal Carcinoma In Situ to Invasive Ductal Carcinoma.

IntroductionBreast atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) are precursor stages of invasive ductal carcinoma (IDC). This study aimed to investigate the pathogenesis of breast cancer by dynamically analyzing expression changes of hub genes from normal mammary epithelium (NME) to simple ductal hyperplasia (SH), ADH, DCIS, and finally to IDC.MethodsLaser-capture microdissection (LCM) data for NME, SH, ADH, DCIS, and IDC cells were obtained. Weighted gene co-expression network analysis (WGCNA) was performed to dynamically analyze the gene modules and hub genes associated with the pathogenesis of breast cancer. Tissue microarray, immunohistochemical, and western blot analyses were performed to determine the protein expression trends of hub genes.ResultsTwo modules showed a trend of increasing expression during the development of breast disease from NME to DCIS, whereas a third module displayed a completely different trend. Interestingly, the three modules displayed inverse trends from DCIS to IDC compared with from NME to DCIS; that is, previously upregulated modules were subsequently downregulated and vice versa. We further analyzed the module that was most closely associated with DCIS (p=7e−07). Kyoto Gene and Genomic Gene Encyclopedia enrichment analysis revealed that the genes in this module were closely related to the cell cycle (p= 4.3e–12). WGCNA revealed eight hub genes in the module, namely, CDK1, NUSAP1, CEP55, TOP2A, MELK, PBK, RRM2, and MAD2L1. Subsequent analysis of these hub genes revealed that their expression levels were lower in IDC tissues than in DCIS tissues, consistent with the expression trend of the module. The protein expression levels of five of the hub genes gradually increased from NME to DCIS and then decreased in IDC. Survival analysis predicted poor survival among breast cancer patients if these hub genes were not downregulated from DCIS to IDC.ConclusionsFive hub genes, RRM2, TOP2A, PBK, MELK, and NUSAP1, which are associated with breast cancer pathogenesis, are gradually upregulated from NME to DCIS and then downregulated in IDC. If these hub genes are not downregulated from DCIS to IDC, patient survival is compromised. However, the underlying mechanisms warrant further elucidation in future studies.

Altered expression of ERK, Cytochrome-c, and HSP70 triggers apoptosis in Quinacrine-exposed human invasive ductal carcinoma cells

Invasive ductal carcinoma (IDC) is the most recurrent cancer, accounting for 80% of all breast cancers worldwide. Originating from the milk duct, it eventually invades the fibrous tissue of the breast outside the duct, proliferation takes 1–2 months for each division. Quinacrine (QC), an FDA-approved small molecule, has been shown to have anti-cancer activity in numerous cancerous cell lines through diverse pathways; ultimately leading to cell death. Here, we have investigated the mode of action of QC in MCF7 cells. This study demonstrated the modulation of cellular cytoskeleton, such as the formation of distinct filopodial and lamellipodial structures and spikes, through the regulation of small-GTPases. We also observed that QC induces a signaling cascade by inducing apoptotic cell death by increasing ROS generation and altering HSP70 expression; which presumably involves ERK regulation. Our findings show that QC could be an attractive chemotherapeutic agent having a “shotgun” nature with potential of inducing different signaling pathways leading to apoptotic cell death. This opens new avenues for research on developing QC as an effective therapeutic agent for the treatment of invasive ductal carcinomas.

Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Coregulator in Invasive Lobular Carcinoma of the Breast.

Invasive lobular carcinoma (ILC) is the most common special histologic subtype of breast cancer, and nearly all ILC tumors express estrogen receptor alpha (ER). However, clinical and laboratory data suggest ILC are strongly estrogen-driven but not equally antiestrogen-sensitive. We hypothesized ILC-specific ER coregulators mediate ER functions and antiestrogen resistance in ILC, and profiled ER-associated proteins by mass spectrometry. Three ER+ ILC cell lines (MDA MB 134VI, SUM44PE, and BCK4) were compared with ER+ invasive ductal carcinoma (IDC) line data, and we examined whether siRNA of identified proteins suppressed ER-driven proliferation in ILC cells. This identified mediator of DNA damage checkpoint 1 (MDC1), a tumor suppressor in DNA damage response (DDR), as a novel ER coregulator in ILC. We confirmed ER:MDC1 interaction was specific to ILC versus IDC cells, and found MDC1 knockdown suppressed ILC cell proliferation and tamoxifen resistance. Using RNA-sequencing, we found in ILC cells MDC1 knockdown broadly dysregulates the ER transcriptome, with ER:MDC1 target genes enriched for promoter hormone response elements. Importantly, our data are inconsistent with MDC1 tumor suppressor functions in DDR, but suggest a novel oncogenic role for MDC1 as an ER coregulator. Supporting this, in breast tumor tissue microarrays, MDC1 protein was frequently low or absent in IDC, but MDC1 loss was rare in ER+ ILC. ER:MDC1 interaction and MDC1 coregulator functions may underlie ER function in ILC and serve as targets to overcome antiestrogen resistance in ILC. IMPLICATIONS: MDC1 has novel ER coregulator activity in ILC, which may underlie ILC-specific ER functions, estrogen response, and antiestrogen resistance.

"Switch of E-Cadherin to N-Cadherin expression in different molecular subtypes of breast invasive duct carcinomas and its correlation with clinicopathological features".

In breast cancer, metastasis and recurrence is the main culprit in treatment failure. This study aimed to explore the role of E-cadherin/N-cadherin Switch in progression, spread and metastasis in breast invasive duct carcinoma. A cross-sectional study on 118 formalinfixed paraffinembedded mastectomy specimens of invasive breast duct carcinoma. Primary antibodies for E-cadherin (monoclonal, clone HECD-1; Zymed Laboratories; dilution 1:600) and N-cadherin (monoclonal, clone 3B9; Zymed Laboratories, Inc., Montrouge, France; dilution 1:200) were applied for all cases. The study revealed that E-cadherin high expression was significantly associated with advanced TNM clinical stage (P = 0.021), and nodal metastasis (P < 0.001). High expression of N-cadherin was significantly positively correlated with tumor sizes (P < 0.00), advanced clinical stage (P < 0.00), and nodal metastasis (P < 0.008). Mean OS was 39.99 months in cases with negative expression versus 41.8 months in cases with positive expression. Mean DFS in cases with positive E. cadh expression was 41.89 months was higher than mean DFS in cases with negative E. cadh expression which was 40.52 months, but it showed no statistical significance (P = 0.57). This study demonstrated that loss of E-cadherin and gain of N-cadherin promotes invasion, migration, and metastasis in invasive ductal carcinoma cells. Importantly, these findings may exploit new cancer therapies using N-cadherin antagonists.

Radiosensitization induced by zinc‐doped hydroxyapatite nanoparticles in breast cancer cells

AbstractZinc‐doped hydroxyapatite (HA) nanoparticles were synthesized by microwave assisted method and used with ionizing radiation for inhibition of proliferation of breast cancer cells. Zinc‐doped HAs were produced in four different compositions in order to determine the best doping rate in terms of physical and biological properties. Nanoparticle characterizations were performed with X‐ray diffractometer, Fourier transform infrared spectroscopy, scanning electron microscopy, and inductively coupled plasma‐mass spectrometry. Viability of MDA‐MB‐231(isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma) cells treated with nano‐HA particles and radiation were assessed by MTT assay. Caspase‐7 and Poly (ADP‐ribose) polymerase protein expressions in samples were examined by the Western blot. X‐ray diffraction patterns of our samples were found to be in good correlation with the reference HA peaks. Notably, increasing zinc amount resulted in elevated percentage of β‐tricalcium phosphate, phases. All formulations including pure HA particles were non‐cytotoxic in MDA MB 231 cells. On the other hand, low rate Zn‐doped HA particles showed significant anti‐proliferation effect during irradiation. The combination of irradiation with Zn‐doped HA particles also induced apoptosis, demonstrated as cleavage of caspase‐7 and PARP proteins. In conclusion, low rate Zn‐doped HA enhanced the radiation effect on breast cancer cells.

Classification of Breast Cancer Cell Images using Multiple Convolution Neural Network Architectures

Breast cancer is a malignant tumor that affects women. It is the most prevalent cancer in women, affecting about 10% of all women at any point in their lives. The development of breast cancer begins in the lobules or ducts of the cells. Early detection and prevention are the best ways to stop this cancer from spreading. In this study, five Convolution Neural Network (CNN) models are used to process image data of breast cells. AlexNet, InceptionV3, GoogLeNet, VGG19 and Xception models are used for the classification of Invasive Ductal Carcinoma, IDC and Non-Invasive Ductal Carcinoma (Non-IDC) cells. The models are trained and tested at different epochs to record the learning rate. It is observed from the study that with higher epochs, the data loss decreases and accuracy increases. The accuracy of InceptionV3 and Xception is 92.48% and 90.72% respectively. Likewise, VGG19 and AlexNet have fairly close accuracy of 94.83% and 96.74%. However, GoogLeNet dominates over the other implemented models with the highest accuracy of 97.80%. The GoogLeNet model performs with high accuracy and precision in detecting IDC cells responsible for breast cancer.